Distinct gene expression patterns of SOX2 and SOX2OT variants in different types of brain tumours.

Numerous investigations have been recently published on the dysregulated expression of long-noncoding RNAs (lncRNAs) in various cancer types, emphasizing that abnormal lncRNA expression is a major contributor to tumourigenesis. A broad spectrum of lncRNAs is expressed in the central nervous system, where these RNAs seem to play key roles in brain development and function. In addition to expressing SOX2, a master regulator of pluripotency that lies within its third intron, lncRNA SOX2OT has a proposed role in regulating neural development. Based on our previous studies, alternative splicing of SOX2OT generates two alternatively spliced variants (SOX2OT-S1 and SOX2OT-S2). The present study investigated the expression patterns of SOX2OT variants and SOX2 in three principal types of brain tumours (gliomas, meningiomas and pituitary adenomas) and in four brain tumour cell lines (U87-MG, 1321N1, A172 and DAOY). Total RNAwas extracted from 34 human brain tumour specimens, and the expression profile of target genes was measured using a real-time reverse transcription PCR approach. Our data revealed distinct expression patterns for SOX2OT variants and SOX2 in the brain tumour samples, indicating their potential involvement in brain tumourigenesis. Moreover, our results highlighted the potential usefulness of SOX2OT-S1, SOX2OT-S2, and SOX2 in molecular diagnosis and brain tumour classification.

Malat1 deficiency prevents neonatal heart regeneration by inducing cardiomyocyte binucleation.

The adult mammalian heart has limited regenerative capacity, while the neonatal heart fully regenerates during the first week of life. Postnatal regeneration is mainly driven by proliferation of preexisting cardiomyocytes and supported by proregenerative macrophages and angiogenesis. Although the process of regeneration has been well studied in the neonatal mouse, the molecular mechanisms that define the switch between regenerative and nonregenerative cardiomyocytes are not well understood. Here, using in vivo and in vitro approaches, we identified the lncRNA Malat1 as a key player in postnatal cardiac regeneration. Malat1 deletion prevented heart regeneration in mice after myocardial infarction on postnatal day 3 associated with a decline in cardiomyocyte proliferation and reparative angiogenesis. Interestingly, Malat1 deficiency increased cardiomyocyte binucleation even in the absence of cardiac injury. Cardiomyocyte-specific deletion of Malat1 was sufficient to block regeneration, supporting a critical role of Malat1 in regulating cardiomyocyte proliferation and binucleation, a landmark of mature nonregenerative cardiomyocytes. In vitro, Malat1 deficiency induced binucleation and the expression of a maturation gene program. Finally, the loss of hnRNP U, an interaction partner of Malat1, induced similar features in vitro, suggesting that Malat1 regulates cardiomyocyte proliferation and binucleation by hnRNP U to control the regenerative window in the heart.

The G3BP1-UPF1-Associated Long Non-Coding RNA CALA Regulates RNA Turnover in the Cytoplasm.

Besides transcription, RNA decay accounts for a large proportion of regulated gene expression and is paramount for cellular functions. Classical RNA surveillance pathways, like nonsense-mediated decay (NMD), are also implicated in the turnover of non-mutant transcripts. Whereas numerous protein factors have been assigned to distinct RNA decay pathways, the contribution of long non-coding RNAs (lncRNAs) to RNA turnover remains unknown. Here we identify the lncRNA CALA as a potent regulator of RNA turnover in endothelial cells. We demonstrate that CALA forms cytoplasmic ribonucleoprotein complexes with G3BP1 and regulates endothelial cell functions. A detailed characterization of these G3BP1-positive complexes by mass spectrometry identifies UPF1 and numerous other NMD factors having cytoplasmic G3BP1-association that is CALA-dependent. Importantly, CALA silencing impairs degradation of NMD target transcripts, establishing CALA as a non-coding regulator of RNA steady-state levels in the endothelium.

The splicing-regulatory lncRNA NTRAS sustains vascular integrity.

Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type-specific manner. By screening for endothelial-enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA-dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO-1) pre-mRNA. Deletion of the hnRNPL binding motif in mice (Ntras∆CA/∆CA ) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α- isoform, which augments permeability of the endothelial monolayer. Ntras∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.

Long non-coding RNAs in vascular biology and disease.

The advent of deep sequencing technologies recently unraveled the complexity of the human genome: Although almost entirely transcribed, only a very minor part of our genome actually accounts for protein coding exons and most is considered non-coding. Among the non-coding transcripts, long non-coding RNAs (lncRNAs) constitute a rather heterogeneous group of linear as well as circular RNAs (circRNAs). LncRNAs act via multiple mechanisms and several lncRNAs were shown to be involved in vascular development, growth and remodeling. For example, the lncRNAs PUNISHER, MALAT1, MEG3, and GATA6-AS regulate vessel formation in vivo, whereas lincRNA-p21 controls smooth muscle cell function and neointima formation. For several other lncRNAs (e.g. SENCR, SMILR, and HypERlnc) functional roles in smooth muscle cells/pericytes have been described in vitro. Less information is available with respect to the function of circRNAs. Here most studies report on expression profiles but some circRNAs (e.g. cANRIL or cZNF292) may also play critical roles in smooth muscle or endothelial cells in vitro. This review summarizes the current knowledge of lncRNA and circRNA functions in vascular biology and disease and discusses their potential use as biomarkers.

Anti-differentiation non-coding RNA, ANCR, is differentially expressed in different types of brain tumors.

Long non-coding RNAs (lncRNAs) are important modulators of various cellular and molecular events, including cancer-associated pathways. The Anti-differentiation ncRNA (ANCR) is a key regulator of keratinocyte differentiation, where its expression is necessary to maintain epidermal progenitor's cells. Herein, we investigated the expression pattern of ANCR in the course of neural differentiation. Moreover, we used published RNAseq data and clinical samples to evaluate the alteration of ANCR expression in different cell types and brain tumors. Furthermore, we manipulated ANCR expression in glioma cell lines to clarify a potential functional role for ANCR in tumorigenesis. Our qRT-PCR results revealed a significant upregulation of ANCR in more malignant and less differentiated types of brain tumors (P = 0.03). This data was in accordance with down regulation of ANCR during neural differentiation. ANCR suppression caused an elevation in apoptosis rate, as well as a G1 cell cycle arrest in glioblastoma cell line. Altogether, our data demonstrated that ANCR may play a role in glioma genesis and that it could be considered as a potential diagnostic and therapeutic target to combat brain cancers.

The lncRNA GATA6-AS epigenetically regulates endothelial gene expression via interaction with LOXL2.

Impaired or excessive growth of endothelial cells contributes to several diseases. However, the functional involvement of regulatory long non-coding RNAs in these processes is not well defined. Here, we show that the long non-coding antisense transcript of GATA6 (GATA6-AS) interacts with the epigenetic regulator LOXL2 to regulate endothelial gene expression via changes in histone methylation. Using RNA deep sequencing, we find that GATA6-AS is upregulated in endothelial cells during hypoxia. Silencing of GATA6-AS diminishes TGF-ß2-induced endothelial-mesenchymal transition in vitro and promotes formation of blood vessels in mice. We identify LOXL2, known to remove activating H3K4me3 chromatin marks, as a GATA6-AS-associated protein, and reveal a set of angiogenesis-related genes that are inversely regulated by LOXL2 and GATA6-AS silencing. As GATA6-AS silencing reduces H3K4me3 methylation of two of these genes, periostin and cyclooxygenase-2, we conclude that GATA6-AS acts as negative regulator of nuclear LOXL2 function.

Novel spliced variants of OCT4, OCT4C and OCT4C1, with distinct expression patterns and functions in pluripotent and tumor cell lines.

OCT4 is a major regulator of pluripotency which has several spliced variants and expressed pseudogenes. Here, we are reporting the existence of two additional novel spliced variants of OCT4, OCT4C and OCT4C1, which lack Exon1 (E1) but start at a novel exon (E0) located ∼14kb upstream of E2. OCT4C/C1 is highly expressed in ES and iPS cells, and their expression was sharply turned off, upon the induction of neural differentiation. The long non-coding RNA (lncRNA) PSORS1C3, is located ∼9kb downstream of the E0 of OCT4C/C1. PSORS1C3 is vigorously spliced to generate nine novel variants, however, none of its exons incorporated in alternatively spliced variants of OCT4. Interestingly, the exons of OCT4 and PSORS1C3 are intertwined, with a novel exon (E0) of PSORS1C3 located ∼4kb upstream of OCT4 E0. This exon participates in generating some more variants of PSORS1C3 (variants 10-24). OCT4C/C1 knock-down in ES and iPS cell lines caused a slight downregulation of PSORS1C3 and OCT4A, a slight upregulation of OCT4B1, and a dramatic upregulation of OCT4B. Altogether, our data revisited the current view of OCT4 gene structure and regulation, and revealed its complex genomic features and expression regulation in stem and tumor cells.

Two novel splice variants of SOX2OT, SOX2OT-S1, and SOX2OT-S2 are coupregulated with SOX2 and OCT4 in esophageal squamous cell carcinoma.

Long noncoding RNAs (lncRNAs) have emerged as new regulators of stem cell pluripotency and tumorigenesis. The SOX2 gene, a master regulator of pluripotency, is embedded within the third intron of a lncRNA known as SOX2 overlapping transcript (SOX2OT). SOX2OT has been suspected to participate in regulation of SOX2 expression and/or other related processes; nevertheless, its potential involvement in tumor initiation and/or progression is unclear. Here, we have evaluated a possible correlation between expression patterns of SOX2OT and those of master regulators of pluripotency, SOX2 and OCT4, in esophageal squamous cell carcinoma (ESCC) tissue samples. We have also examined its potential function in the human embryonic carcinoma stem cell line, NTERA2 (NT2), which highly expresses SOX2OT, SOX2, and OCT4. Our data revealed a significant coupregulation of SOX2OT along with SOX2 and OCT4 in tumor samples, compared to the non-tumor tissues obtained from the margin of same tumors. We also identified two novel splice variants of SOX2OT (SOX2OT-S1 and SOX2OT-S2) which coupregulated with SOX2 and OCT4 in ESCCs. Suppressing SOX2OT variants caused a profound alteration in cell cycle distribution, including a 5.9 and 6.9 time increase in sub-G1 phase of cell cycle for SOX2OT-S1 and SOX2OT-S2, respectively. The expression of all variants was significantly diminished, upon the induction of neural differentiation in NT2 cells, suggesting their potential functional links to the undifferentiated state of the cells. Our data suggest a part for SOX2OT spliced variants in tumor initiation and/or progression as well as regulating pluripotent state of stem cells.